Efflux Pump-Mediated Drug Resistance in Candida krusei

Severely immunocompromised individuals often suffer from oesophageal and oropharyngeal candidosis. The yeast Candida krusei is emerging as a significant pathogen in these patients due to its innate fluconazole resistance that may be caused by drug efflux mechanisms. Objectives: The aim of this study was to clone the CkABC1 gene from C. krusei and hyper-express the ATP-binding cassette (ABC) protein CkAbc1p in the model yeast Saccharomyces cerevisiae in order to study its possible pump function. Methods: The CkABC1 gene was isolated from genomic DNA (gDNA) by inverse PCR. The CkABC1 genes from both gDNA and cDNA were cloned in vector pABC3 downstream of the S. cerevisiae PDR5 promoter. A cassette removed from recombinant pABC3 containing CkABC1 and the URA3 gene was used to transform S. cerevisiae strain AD1-8u- to Ura+ by integration at the PDR5 locus. The function of the CkAbc1p was investigated by measuring the susceptibility of S. cerevisiae transformants to antifungal drugs and their ability to efflux the fluorescent pump substrate rhodamine 6G (R6G). Results: The CkABC1 open reading frame was 4654 bp long and contained an intron of 88 bp that was confirmed by sequencing its cDNA. CkAbc1p (1521 amino acids) shared between 52% and 58% identity with ABC transporters from related fungi such as Candida glabrata, Candida albicans, and Candida dubliniensis, known to be involved in azole drug resistance. Disk diffusion assays confirmed that CkAbc1p was functionally expressed from both gDNA and cDNA and conferred on S. cerevisiae resistance to several azole antifungals. Conclusions: CkAbc1p is homologous to other fungal ABC drug pumps. It pumped a range of compounds and may be involved in the innate resistance of C. krusei to azole antifungals. This work was supported by an IADR David B. Scott Fellowship awarded to A. Ranchod.